Evaluation of common protein biomarkers involved in the pathogenesis of respiratory diseases with proteomic methods: A systematic review

Abstract Aim Respiratory disease (RD) is one of the most common diseases characterized by lung dysfunction. Many diagnostic mechanisms have been used to identify the pathogenic agents of responsible for RD. Among these, proteomics emerges as a valuable diagnostic method for pinpointing the specific proteins involved in RD pathogenesis. Therefore, in this study, for the first time, we examined the protein markers involved in the pathogenesis of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), asthma, bronchiolitis obliterans (BO), and chemical warfare victims exposed to mustard gas, using the proteomics method as a systematic study. Materials and Methods A systematic search was performed up to September 2023 on several databases, including PubMed, Scopus, ISI Web of Science, and Cochrane. In total, selected 4246 articles were for evaluation according to the criteria. Finally, 119 studies were selected for this systematic review. Results A total of 13,806 proteins were identified, 6471 in COPD, 1603 in Asthma, 5638 in IPF, three in BO, and 91 in mustard gas exposed victims. Alterations in the expression of these proteins were observed in the respective diseases. After evaluation, the results showed that 31 proteins were found to be shared among all five diseases. Conclusion Although these 31 proteins regulate different factors and molecular pathways in all five diseases, they ultimately lead to the regulation of inflammatory pathways. In other words, the expression of some proteins in COPD and mustard‐exposed patients increases inflammatory reactions, while in IPF, they cause lung fibrosis. Asthma, causes allergic reactions due to T‐cell differentiation toward Th2.


| INTRODUCTION
Respiratory disease (RD) is one of the leading causes of patient mortality worldwide.2][3] A wide range of clinical symptoms is observed in RD patients according to the type of disease and its pathogenesis.The pathogenesis of RD is not fully understood; however, several mechanisms and factors have been implicated in their development and progression. 1,4Inflammation, coagulation, and oxidative stress pathways are known to play important roles in the development and progression of RDs.Studies have shown that some of these pathways, including inflammatory pathways and oxidative stress, are involved in developing COPD and Asthma.Activation of the NF-κB pathway in COPD has been shown to lead to inflammation.][7][8][9] Based on the evidence obtained, inflammation is the most important pathway in the pathogenesis of RD.Most cells in the respiratory pathway induce the production of cytokines.On the other hand, factors produced by respiratory cells lead to the production of inflammatory mediators that result in the chemotaxis of immune cells.Most oxidative stress pathways lead to inflammation.][12] Pulmonary disorders have also been shown in patients exposed to mustard gas and idiopathic pulmonary fibrosis (IPF) due to the activation of inflammatory pathways. 13So far, common factors and pathways involved in the pathogenesis of RD have not been identified.It has also been found that RD, including COPD, has not been correctly diagnosed in many cases.Spirometry testing is not practical for diagnosing and differentiating COPD from other RD.Including warfare victims exposed to mustard gas, some patients have inflammation in the airways, similar to Asthma in terms of clinical symptoms.5][16] Therefore, there is an urgent need for efficient diagnostic biomarkers to differentiate between these illnesses and common indicators.
Various methods have been used to identify markers, but it has been shown that proteomics can help locate involved features in RD pathogenesis; it is a highthroughput method for biomarker panel discovery with high sensitivity and specificity.8][19] Proteomics is an approach which dramatically has been used in recent decades to detect proteins in many diseases.Also, new proteins identified by this method can be effective in early diagnosis, follow-up, and patient management.Identifying common protein markers in RD can be a suitable solution for designing target therapy and preventive strategies for RD patients. 18urthermore, since inflammation is recognized as a primary pathogenesis pathway in RD, the identification of biomarkers associated with it can facilitate easier management of patients.On the other hand, the identification of factors associated with inflammatory pathways can be helpful in patient management.
No study has been performed to identify protein markers commonly involved in RD pathogenesis.Also, most studies have examined the protein markers involved in the pathogenesis of RD; the results of many of them are challenging.
Therefore, for the first time in this study, we examined the protein markers involved pathogenesis of COPD, IPF, Asthma, bronchiolitis obliterans (BO), and chemical warfare victims exposed to mustard gas using the proteomics method as a systematic study.Finding shared proteins among these respiratory disorders is the goal of this research, which will also help create common therapeutic approaches and a better understanding of disease mechanisms.

| RESULTS
This study examined five diseases of COPD, Asthma, IPF, BO, and mustard gas-exposed victims.In total, 4946 articles entered endnote X7 software.After deleting the duplicates and other articles, reviewed the full text of 148 articles based on the reasons mentioned in Figure 1.Finally, 119 studies were selected in this systematic review.Details and related findings of selected papers (age, sex, sample type, type of technique used, statistical analysis, number of increased or decreased proteins, model characteristics, validation test) are shown in supplementary files.  It lied for COPD, IPF, Asthma, BO, and mustard gas-exposed victims, respectively.

| Biological specimens
Using different samples in 119 selected studies.Accordingly, in COPD, the most commonly used selection was peripheral blood (PB); serum, plasma, or mononuclear cells were also isolated (25 of 56 studies). 114,119,123 Also sed were saliva samples in one study.50,63,115,117,124 In one study, the exosome was used; in another, one nasal brush was used as a sample.8,121 BALF was used in one study, and tissue as a biological sample in another.BALF was used in BO in two studies. 20,21

| Technological platform used
Proteomics is a technique that evaluates the proteins in the samples; they can be cells, solutions, tissues, and many different biological samples.Proteomics includes other techniques depending on the sample type and the study's purpose. 125In the studies included in the systematic review, various methods have been used.In COPD and IPF, the most commonly used techniques were Two-dimensional gel electrophoresis (2DE) and liquid chromatography with tandem mass spectrometry (LC-MS-MS).In Asthma, LC-MS-MS was used more than any other technique (13 out of 27).Matrix-assisted laser desorption/ionization (MALDI) coupled with time-of-flight (MALDI-TOF) was more commonly used in BO (Supporting Information S1).In mustard gas-exposed victims, four studies used 2DE, and 5 out of 7 studies used MALDI-TOS-MS/MS.Most studies used western blot and enzyme-linked immunosorbent assay (ELISA) techniques for validation (Supporting Information S1).

| Proteomic biomarkers
After selecting the articles investigated, the expression changes of proteins were identified by proteomics and, in COPD, altered the expression of 6471 proteins.Asthma changed the face of 1603 proteins.Also, in IPF, 5638 proteins, and BO and mustard gas exposed victims, their 3 and 91 proteins expression were altered, respectively.Alteration of protein expression, or increase in their face in five diseases according to the control group, was considered.
In this study, after removing duplications, identified 13,806 proteins in five diseases.After evaluation, 31 proteins were present in five conditions in the results section.After evaluating 31 proteins, we found that most are involved in the pathogenesis of inflammation and coagulation.Biological properties and their functions under normal conditions inside the cell are listed in Table 1. 164 Inflammion is one of the primary pathogens of RD; most of the 31 common proteins in cited diseases were involved in inflammatory reactions.C3 is one of the components of complement that plays a vital role in the immune system, especially innate immunity.Studies have shown that C3 expression increases in some diseases, especially COPD and chemical warfare victims exposed to mustard gas. 165,166C3 activates the NF-κB pathway, which ultimately triggers the nucleotide- binding oligomerization domain, leucine-rich repeat, and pyrin domain containing (NLRP); it also induces inflammatory cytokines production, including IL-1.It has also been shown that increased C3 expression in COPD leads to netosis.
Netosis is when neutrophils transfer their contents outward, creating a network that leads to the accumulation of immune cells and inflammation.Netosis increases TLR4 expression, which ultimately increases platelet activation.][169] Increased ROS production causes neutrophil death and ketosis.Increased production of inflammatory cytokines can differentiate T helper2 (Th2) cells, which produce IL-13 and IL-5, leading to allergic reactions in patients, especially asthma patients. 168n the other hand, C3 has been shown to inhibit the lipoxin A4 (LXA4) expression by activating the MAPK/ERK pathway.LXA4 has been shown to regulate pulmonary epithelial cells by regulating tight junctions and preventing inflammation.1][172][173][174] TF is the starting factor in the coagulation cascade, the word of which can cause thrombosis.Increased C3 expression in IPF leads to activation of the TGF-β/SMAD pathway, which ultimately causes lung fibrosis. 175,176herefore, targeting C3 in mentioned diseases can prevent inflammation and disease progression.
Apolipoprotein A-1 (ApoA1) is a component of HDL that is involved in cardiovascular disease prevention by regulating blood cholesterol levels.However, ApoA1 has also been shown to have an anti-inflammatory role.It acts as a protective agent in preventing the progression of many diseases, including RD.In Asthma, APOA1 increases TGF-β expression.Studies have shown that TGF-β causes inflammation and airway obstruction by activating actin in epithelial cells. 177][180][181] However, ApoA1 in IPF has been shown to prevent inflammation by inhibiting TGF-β expression.ApoA1 also inhibits inflammation and inflammatory cytokines production by inhibiting NF-κB.In COPD, APOA1 expression has been shown to reduce the production of inflammatory cytokines, including IL-1 and TNF-α. 182iven that APOA1 suppresses NF-κB can hypothesize that in COPD, APOA1 may reduce inflammatory cytokines production and inhibit inflammation by suppressing NF-κB expression.
NF-κB expression increases ferritin expression.Ferritin is one of the acute phase proteins that consists of two light and heavy chains.Asthma has been shown to reduce the expression of heavy-chain ferritin (FTH1), which increases inflammation and oxidative stress.Therefore, it has been demonstrated that MAPK/ERK/ c-jun pathway increases FTH1 expression. 183However, this pathway is suppressed in Asthma.][186] Studies have shown that increased ferritin expression in IPF, unlike Asthma, is associated with a poor prognosis leading to increased pulmonary fibrosis. 187It has also been shown that increased ferritin expression in COPD can be related to airway obstruction and disease progression (Figure 2). 188ransferrin (TF) is another factor that plays a vital role in the pathogenesis of RD, the same as ferritin.Accordingly, it has been shown that in asthma and COPD, increased TF expression leads to TLR2 activation and, ultimately, inflammation.CD71 is known as the TF receptor.Heat shock protein (HSP) in COPD reduces inflammation by inhibiting the CD71/JNK/NF-κB pathway.HSP also increases keratin expression and inflammation through the EGF/AKT pathway.In asthma, HSP causes inflammation by activating the ERK1/2/NF-κB pathway.][191][192][193][194] While shown in the mustard gas-exposed patient, TF has a protective role.
TF reduces ROS production and inflammation.Therefore, can hypothesize that TF may prevent cell damage in patients by expressing Nrf2, which has an antioxidant role. 195On the other hand, HSP has been shown to cause pulmonary fibrosis by activating the TGF-β/SMAD pathway. 196,197HSP acts as a double-edged sword and plays an influential role in the occurrence and inhibition of inflammation; it is possible to control the progression of inflammation and improve patient survival by identifying its upstream and downstream pathways (Figure 2).Lipocalin1 (LCN1) is another molecule like TF; it has a protective role.In COPD, LCN1 expression increased, associated with inflammation and oxidative stress prevention.LCN1 has been shown to increase the expression of Nrf2 and HO-1 by activating the MAPK/ ERK pathway.These two factors are antioxidants and prevent cell damage. 198LCN1, like COPD, plays a protective role in the mustard gas-exposed patient and protects cells against ROS. 199,200In contrast, increased LCN1 expression in asthma has been shown to activate the NF-κB pathway and causes inflammation. 201In IPF, increased LCN1 expression activates the NF-κB pathway through MMP-9.However, LCN1 expression in IPF is reduced by Peroxisome proliferator-activated receptor gamma (PPAR-γ) (Figure 2). 202etinol binding protein 4 (RBP4) is one of the genes involved in the pathogenesis of RD and regulates TGF-β.In COPD, decreased RBP4 expression is associated with increased disease progression.Albumin (ALB) stabilizes by binding to RBP4.ALB/RBP4 then reduces inflammation by inhibiting NF-κB.In addition, it blocks the TGFβ/SMAD signaling pathway. 203In asthma, decreased RBP4 expression is associated with increased inflammation. 204In IPF, TGF-β increases the expression of proteasome 20S subunit alpha 2 (PSMA2), leading to inflammation and pulmonary fibrosis.In asthma, PSMA2 expression causes inflammation by activating the NF-κB and producing inflammatory cytokines. 205In COPD, PSMA2 activates NF-κB and caspase3, causing lung cell apoptosis and worsening patients' clinical course.Thus, targeting RBP4 prevents inflammation and reduces apoptosis (Figure 2). 206 general, in RDs, increased expression of inflammatory genes causes exacerbation of disease.Activation of NF-κB lead to the production of cytokine by immune cell and induce activation of inflammation signaling pathway.][209][210] The strengths of this study are as follows: This study focuses on the proteomic analysis of five diseases, namely COPD, IPF, asthma, chemical warfare victims exposed to mustard gas, and BO.It adopts a systematic review approach, which is the first of its kind.Furthermore, common proteins among the investigated diseases have been identified, which can be potentially utilized in the future for designing appropriate therapeutic strategies.
The limitation of the study are as follows: The methods used in the reviewed articles mostly consist of high dropout.Also, the study did not include an experimental validation of the identified proteins, which would provide more evidence for their potential role in disease pathogenesis.Considering that 31 proteins (TF, GSTP1, UBE2N, HP, EIF5A, KRT9, APO A4, ALB, APO C1, ANG, S100A8, FGA, HSPB1, C3, LCN1, CRP, FCN2, ADIPOQ, IGKC, MPO, PSMA2, ACTB, S100A9, GC, FTH1, AHSG, GAPDH, APO C3, PPIA, RBP4, and KRT10) are common in five diseases, identifying their upstream and downstream pathways can be essential in designing treatment strategies and managing patients.

| FUTURE PERSPECTIVE
It would be better to conduct experimental studies on human samples to confirm the results of this study.Additionally, examining differential proteins in each of the diseases and conducting additional studies on other OMICs, including transcriptome and metabolome, and integrating their data with proteome, can provide a better understanding of the conditions.
B. Fatemeh Nobakht M. Gh has conceived the manuscript and revised it.Hadi Rezaeeyan, B. Fatemeh Nobakht M. Gh, and Masoud Arabfard extraction data and analysis.Hamid R. Rasouli and Alireza Shahriary design strategy search.All authors read and approved the final manuscript.